Lactic acid bacteria such as Lactobacillus plantarum and Oenococcus oeni are used in the wine industry for malolactic fermentation. Their functionality is based on the ability to convert malic acid into the gentler lactic acid which is used for many types of wine. Over the last decades, average temperatures have risen noticeably in the wine producing countries leading to ripened grapes with higher sugar content thereby producing wines with higher alcohol. Today many wines have an alcohol content of 14-15% and this is a substantial challenge and stress condition for the fermenting microorganism such as yeast and lactic acid bacteria.
As discussed in U.S. Pat. No. 7,625,745 B2 (Danstar Ferment, CH)—in traditional winemaking, the malolactic fermentation (MLF) is produced by means of the spontaneous growth of an indigenous flora of lactic acid bacteria. The process of MLF begins of its own accord, when the malolactic flora is sufficiently developed, that is to say in a random manner between the end of alcoholic fermentation and several weeks, even several months, after the alcoholic fermentation. When the malolactic bacteria reach a concentration of about 106 CFU/ml in the medium, they enter an active metabolic phase and start the fermentation of the malic acid. In these conditions, Oenococcus oeni is the species most frequently responsible for the MLF. In fact, if at the start of alcoholic fermentation a predominance of the homofermentative Lactobacillus plantarum and Lactobacillus casei species is observed, these disappear when the alcohol content increases. After alcoholic fermentation, it is the species Pediococcus and Oenococcus, depending on the pH, which predominate and finally reach the critical concentration to start the MLF.
In short, one may say that natural/wildtype Lactobacillus plantarum strains have a relatively low inherent resistance to the concentrations of ethanol/alcohol present in the grape juice during wine production.
U.S. Pat. No. 7,625,745 B2 (PCT filed 2004 and published in 2009) describes the selection of alcohol-resistant Lactobacillus plantarum lactic acid bacterial strains and it is said that the authors believed that it was unexpected that it was possible to select such alcohol-resistant Lactobacillus plantarum lactic acid bacterial strains—e.g. due to that this resistance to alcohol was hitherto unknown for such the Lactobacillus plantarum strains (see e.g. C4, I. 20-30 and figures of U.S. Pat. No. 7,625,745 B2).
It is here relevant to note that in U.S. Pat. No. 7,625,745 B2 a screening was made of natural Lactobacillus plantarum strains arising from fermented wines (see e.g. Example 1 of U.S. Pat. No. 7,625,745 B2).
The isolates of natural lactic acid bacteria were subjected to a selection pressure of resistance to alcohol levels above 10% and two particular L. plantarum strains were found to be sufficiently resistant to alcohol levels above 10% (see e.g. Example 7 of U.S. Pat. No. 7,625,745 B2).
D-cycloserine (D-4-amino-isoxazolidone) is an antibiotic which inhibits alanine racemase, D-alanyl-D-alanine ligase, D-alanylalanine synthase and D-alanine permease causing cell lysis.
D-alanine racemase is essential for the production of D-alanine, an integral part of the peptidoglycan layer of the cell wall.
Strains of Lactobacillus plantarum in which the alanine racemase gene (air) of Lactococcus lactis has been inserted on a plasmid have resistance to D-cycloserine (Bron et al., 2002 Appl Environ Microbiol. 68:5663-5670).
It is here relevant to note that above discussed article of Bron et al does not in any way relate to identification of Lactobacillus plantarum strains with improved resistance to high concentrations of ethanol.
To the knowledge of the present inventors—there is in the prior art not described or suggested any herein relevant link between increased resistance to D-cycloserine and improved resistance to high concentrations of ethanol.